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Charlotte "Charlie" Newton is a bored teenager living in the idyllic town of Santa Rosa, California. She receives wonderful news: her mother's younger brother (her namesake), Charlie Oakley, is arriving for a visit. Two men appear, supposedly working on a national survey. One takes a photo of Uncle Charlie, who demands the roll of film because "no one takes my photograph." The younger surveyor, Jack Graham, asks young Charlie out, and she guesses that he is really a detective. He explains that her uncle is one of two suspects who may be the "Merry Widow Murderer". Charlie refuses to believe it at first, but then observes Uncle Charlie acting strangely. The initials engraved inside a ring he gave her match those of one of the murdered women. During a family dinner he reveals his hatred of rich widows, comparing them to fat animals deserving to be slaughtered.

One night, when Charlie's father and Herbie discuss how to commit the perfect murder, Uncle Charlie lets his guard down and describes elderly widows as "fat, wheezing animals"; he then says, "What happens to animals when they get too fat and too old?" Horrified, Charlie runs out. Uncle Charlie follows and takes her into a seedy bar. He admits he is one of the two suspects. He begs her for help; she reluctantly agrees not to say anything, as long as he leaves soon, to avoid a horrible confrontation that would destroy her mother, who idolizes her younger brother. Detective Saunders tells Charlie that the photo they took of Uncle Charlie was sent for identification by witnesses. News breaks that an alternative suspect was chased by police and killed by an airplane propeller; it is assumed that he was the murderer. Jack tells young Charlie that he loves her and would like to marry her, and leaves.

Uncle Charlie is delighted to be exonerated, but young Charlie knows all his secrets. Soon, she falls down dangerously steep stairs which were cut through. Uncle Charlie says he wants to settle down, and young Charlie says she will kill him if he stays. Later that night, she is trapped in the garage with a car spewing exhaust fumes, and almost dies.

Uncle Charlie announces he is leaving for San Francisco, along with a rich widow, Mrs. Potter. Young Charlie boards the train with her younger sister Ann and their brother to see Uncle Charlie's stateroom. As the children disembark, Uncle Charlie restrains his niece Charlie on the train, hoping to kill her by shoving her out after it picks up speed. In the ensuing struggle, he falls in front of an oncoming train. At his funeral, Uncle Charlie is honored by the townspeople. Jack has returned, and Charlie confesses that she withheld crucial information. They resolve to keep Uncle Charlie's crimes secret.

Alfred Hitchcock appears about 16 minutes into the film, on the train to Santa Rosa, playing bridge with a man and a woman (Dr. and Mrs. Harry). Charlie is traveling on the train under the assumed name of Otis. Mrs. Harry is eager to help Otis, who is feigning illness in order to avoid meeting fellow passengers, but Dr. Harry is not interested and keeps playing bridge. Dr Harry replies to Hitchcock that he doesn't look well while Hitchcock is holding a full suit of spades, the best hand for bridge.

Shadow of a Doubt was both filmed and set in Santa Rosa, California, which was portrayed as a paragon of a supposedly peaceful, small, pre-War American city. Since Thornton Wilder wrote the original script, the story is set in a small American town, a popular setting of Wilder's, but with an added Hitchcock touch to it. In Patrick McGilligan's biography of Hitchcock, he said the film was perhaps the most American film that Hitchcock had made up to that time.

The Newton family home is located at 904 McDonald Avenue in Santa Rosa, which is still standing. The stone railway station in the film was built in 1904 for the Northwestern Pacific Railroad and is one of the few commercial buildings in central Santa Rosa to survive the earthquake of April 18, 1906. The station is currently a visitor center. Some of the buildings in the center of Santa Rosa that are seen in the film were damaged or destroyed by earthquakes in 1969; much of the area was cleared of debris and largely rebuilt. The library was a Carnegie Library which was demolished in the mid-1960s due to seismic concerns.

The film was scored by Dimitri Tiomkin, his first collaboration with Hitchcock (the others being Strangers on a Train, I Confess and Dial M for Murder). In his score, Tiomkin quotes the famous Merry Widow Waltz of Franz Lehár, often in somewhat distorted forms, as a leitmotif for Uncle Charlie and his serial murders. During the opening credits, the waltz theme is heard along with a prolonged shot of couples dancing.

Upon release, the film received unanimously positive reviews. New York Times critic Bosley Crowther loved the film, stating that "Hitchcock could raise more goose pimples to the square inch of a customer's flesh than any other director in Hollywood".[2]Time Magazine called the film "superb"[2] while Variety stated that "Hitchcock deftly etches his small-town characters and homey surroundings".[2]

In a 1964 interview on Telescope with host Fletcher Markle, Markle noted, "Mr Hitchcock, most critics have always considered Shadow of a Doubt, which you made in 1943, as your finest film." Hitchcock replied immediately, "Me too." Markle then asked, "That is your opinion of it still?" Hitchcock replied, "Oh, no question." At the time, Hitchcock's most recent work was Marnie. When later interviewed by François Truffaut, Hitchcock denied the suggestion that Shadow of a Doubt was his "favourite".[3] But in the audio interview with Truffaut, Hitchcock confirmed it was his favourite film, and later reiterated that Shadow of a Doubt was his favorite film in his interview with Mike Douglas in 1969 and in his interview with Dick Cavett in 1972. Alfred Hitchcock's daughter Patricia Hitchcock also said that her father's favorite film was Shadow of a Doubt in the documentary "Beyond Doubt: The Making of Hitchcock's Favorite Film".

Today, the film is still regarded as a masterpiece. Contemporary critic Dave Kehr called it Hitchcock's "first indisputable masterpiece."[4] Many other critics have agreed. Based on 30 reviews on the website Rotten Tomatoes, the film has received 100%, with a consensus of "Alfred Hitchcock's earliest classic -- and his own personal favorite -- deals its flesh-crawling thrills as deftly as its finely shaded characters".[2] When asked by critics as to an overarching theme for the film Hitchcock responded: "Love and good order is no defense against evil". In his book Bambi vs. Godzilla, David Mamet calls it Hitchcock's finest film.